Sorting particles in a microfluidic device

ABSTRACT

A microfluidic device includes a particle sorting region having a first, second and third microfluidic channels, a first array of islands separating the first microfluidic channel from the second microfluidic channel, and a second array of islands separating the first microfluidic channel from the third microfluidic channel, in which the island arrays and the microfluidic channels are arranged so that a first fluid is extracted from the first microfluidic channel into the second microfluidic channel and a second fluid is extracted from the third microfluidic channel into the first microfluidic channel, and so that particles are transferred from the first fluid sample into the second fluid sample within the first microfluidic channel.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 15/891,579, filed on Feb.8, 2018, which is a divisional of U.S. patent application Ser. No.14/931,223, filed on Nov. 3, 2015, which claims the benefit of U.S.Provisional Patent Application No. 62/074,213, filed Nov. 3, 2014 andU.S. Provisional Patent Application No. 62/074,315, filed Nov. 3, 2014,each of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates to particle sorting across microfluidicstreamlines.

BACKGROUND

Particle separation and filtration have been used in numerousapplications across industries and fields. Examples of such applicationsinclude chemical process and fermentation filtration, waterpurification/wastewater treatment, sorting and filtering components ofblood, concentrating colloid solutions, and purifying and concentratingenvironmental samples. Various macro-scale techniques have beendeveloped for use in these applications including methods such ascentrifugation and filter-based techniques. Typically, such techniquesrequire systems that are large, bulky, and expensive and have complexmoving components.

In certain cases, micro-scale techniques offer advantages overmacro-scale techniques, in that scaling down allows the use of uniquehydrodynamic effects for particle sorting and filtration, and thuseliminates the need for large systems with complex moving components.Moreover, micro-scale techniques offer the possibility of portabledevices capable of performing sorting and filtration at much lower costthan larger macro-scale systems. However, typical micro-scale sortingand filtration devices may be limited in the amount of fluid they canhandle over a specified period of time (i.e., low throughput),potentially placing such devices at a disadvantage to their macro-scalecounterparts.

SUMMARY

The present disclosure is based, at least in part, on the discovery thatif one carefully controls the geometries and dimensions of microfluidicdevices one can combine both fluid extraction and inertial lift forcesfor the purpose of sorting and/or shifting particles within or amongfluids. In particular, through fluid extraction and inertial liftforces, the microfluidic devices disclosed herein may be used totransfer fluids to and across different fluidic channels of the device,without an accompanying shift of particles, such that the particles maybe indirectly transferred to another fluid. Alternatively, or inaddition, the techniques disclosed herein can be used in certainimplementations to manipulate not only the transfer of fluids acrossmicro-channels but also the position of particles suspended within afluid sample through the shifting of the particles across fluidstreamlines.

For instance, a first fluid containing particles may be introduced intoa first microfluidic channel having arrays of rigid island structuresseparating the channel from two adjacent microfluidic channels. Fluid isextracted from the first microfluidic channel into one of the adjacentmicrofluidic channels through gaps between island structures in a firstarray, so that the particles are drawn nearer to the island structures.As the particles reach nearer to the island structures, the particlesexperience an inertial lift force away from the direction of fluidextraction such that the particles remain in the first channel. At thesame time, a second fluid from the other adjacent microfluidic channelpasses into the first microfluidic channel through gaps between islandstructures in a second array. As the fluid from the other adjacentchannel enters the first channel, the particles within the first channelcross fluid streamlines, resulting in the shift of the particles fromthe first fluid to the second fluid. If the amount of first fluid thatis extracted from the first channel at each gap of the first arrayequals the amount of second fluid that enters the first channel at eachgap of the second array, then a constant particle concentration can bemaintained.

In addition to shifting particles between fluids, the combination offluid extraction and inertial lift force enables a number of differentways of manipulating fluids and particles. For example, in someimplementations, different types of particles may be separated intodifferent channels, e.g., larger particles may be separated from smallerparticles, to achieve micro-scale sorting of particles and/or filteringof particles from fluids. Alternatively, in some implementations, thecombination of fluid extraction and inertial lift may be used to mixdifferent types of particles. In some cases, both particle separationand shifting between fluids (or particle mixing and shifting betweenfluids) may be performed together. In another example, the combinedfluid extraction and inertial lift forces may be used to focus particlesto desired positions within a microfluidic channel. These and otherapplications may be scaled over large numbers of microfluidic channelsto achieve high throughput sorting/filtering of fluids in systems withlow device fabrication costs.

In general, in one aspect, the subject matter of the present disclosuremay be embodied in a microfluidic device including a particle sortingregion having a first microfluidic channel, a second microfluidicchannel extending along the first microfluidic channel, and a firstarray of islands separating the first microfluidic channel from thesecond microfluidic channel, in which each island in the array isseparated from an adjacent island in the array by an opening thatfluidly couples the first microfluidic channel to the secondmicrofluidic channel, and in which the first microfluidic channel, thesecond microfluidic channel, and the first array of islands are arrangedso that a fluidic resistance of the first microfluidic channel relativeto a fluidic resistance of the second microfluidic channel changes alonga longitudinal section of the particle sorting region, such that aportion of fluid from a fluid sample in the first microfluidic channelor the second microfluidic channel passes through the opening.

In general, in another aspect, the subject matter of the presentdisclosure may be embodied in a microfluidic device that includes: aparticle sorting region having a first microfluidic channel, a secondmicrofluidic channel extending along a first side of the firstmicrofluidic channel, a first array of islands separating the firstmicrofluidic channel from the second microfluidic channel, in which eachisland in the first array is separated from an adjacent island in thefirst array by an opening that fluidly couples the first microfluidicchannel to the second microfluidic channel, a third microfluidic channelextending along a second side of the first microfluidic channel, thesecond side being opposite to the first side of the first microfluidicchannel, a second array of islands separating the first microfluidicchannel from the third microfluidic channel, in which each island in thesecond array is separated from an adjacent island in the second array byan opening that fluidly couples the first microfluidic channel to thethird microfluidic channel, in which the first microfluidic channel, thesecond microfluidic channel, and the first array of islands are arrangedso that the fluidic resistance of the second microfluidic channeldecreases along a longitudinal direction of the particle sorting regionrelative to the fluidic resistance of the first microfluidic channel,such that, during operation of the microfluidic device, a portion offluid from a fluid sample in the first microfluidic channel passesthrough the first array into the second microfluidic channel, and inwhich the first microfluidic channel, the third microfluidic channel andthe second array of islands are arranged so that a fluidic resistance ofthe third microfluidic channel increases along the longitudinaldirection of the particle sorting region relative to the fluidicresistance of the first microfluidic channel, such that, duringoperation of the microfluidic device, a portion of fluid from a fluidsample in the third microfluidic channel passes through the second arrayinto the first microfluidic channel.

Implementations of the devices may have one or more of the followingfeatures. For example, in some implementations, the changing fluidicresistance is a function of an increasing cross-sectional area of thefirst microfluidic channel or the second microfluidic channel along alongitudinal direction of the particle sorting region. For instance, awidth of one of the first microfluidic channel or the secondmicrofluidic channel may increase along the longitudinal direction. Awidth of the other of the first microfluidic channel or the secondmicrofluidic channel may be substantially constant along thelongitudinal direction. Alternatively, a width of the other of the firstmicrofluidic channel or the second microfluidic channel may decreasealong the longitudinal direction.

In some implementations, the decrease in the fluidic resistance of thesecond microfluidic channel relative to the fluidic resistance of thefirst microfluidic channel is a function of an increasingcross-sectional area of the second microfluidic channel along thelongitudinal direction of the particle sorting region. A width of thefirst microfluidic channel can be substantially constant along thelongitudinal direction.

In some implementations, a cross-sectional area of the gaps betweenislands in the first array increases along the longitudinal direction,the cross-sectional area of each gap in the first array being definedalong a plane that is transverse to fluid flow through the gap. Theincrease in the fluidic resistance of the third microfluidic channelrelative to the fluidic resistance of the first microfluidic channel canbe a function of a decreasing cross-sectional area of the thirdmicrofluidic channel along the longitudinal direction of the particlesorting region. A width of the first microfluidic channel can besubstantially constant along the longitudinal direction.

In some implementations, a cross-sectional area of the gaps betweenislands in the second array increases along the longitudinal direction,the cross-sectional area of each gap in the second array being definedalong a plane that is transverse to fluid flow through the gap.

In some implementations, the microfluidic devices further include: afirst inlet channel; and a second inlet channel, in which each of thefirst inlet channel and the second inlet channel is fluidly coupled tothe particle sorting region.

In some implementations, a size of each opening in the first array isgreater than a size of a previous opening in the array along thelongitudinal section.

In some implementations, the particle and fluid shifting region furtherincludes a third microfluidic channel extending along the firstmicrofluidic channel, and a second array of islands separating the firstmicrofluidic channel and the third microfluidic channel, in which thefirst microfluidic channel is located between the second and thirdmicrofluidic channels. The changing relative fluidic resistance may be afunction of an increasing cross-sectional area of the secondmicrofluidic channel or the third microfluidic channel along alongitudinal direction of the particle sorting region. For instance, awidth of one of the second microfluidic channel or the thirdmicrofluidic channel may increase along the longitudinal direction.Alternatively, a width of the other one of the second microfluidicchannel or the third microfluidic channel may decrease along thelongitudinal direction. In some cases, a width of the first microfluidicchannel may be substantially constant along the longitudinal direction.The changing relative fluidic resistance may be a function of anincreasing cross-sectional area of the second microfluidic channel andthe third microfluidic channel along a longitudinal direction of theparticle sorting region.

In some implementations, the devices further include a first inletchannel and a second inlet channel, in which each of the first inletchannel and the second inlet channel is fluidly coupled to the particlesorting region.

In another aspect, the subject matter of the present disclosure may beembodied in methods of sorting particles in a fluid sample, in which themethods include flowing a first fluid sample containing a group of afirst type of particle into a particle sorting region of a microfluidicdevice, in which the particle sorting region includes a firstmicrofluidic channel, a second microfluidic channel extending along thefirst microfluidic channel, and a first array of islands separating thefirst microfluidic channel from the second microfluidic channel. Themethods may further include flowing a second fluid sample into theparticle sorting region, in which a fluidic resistance between the firstmicrofluidic channel and the second microfluidic channel changes along alongitudinal section of the particle sorting region such that a portionof the first fluid sample passes from the first microfluidic channelinto the second microfluidic channel through openings between islands inthe first array, and in which the first microfluidic channel, the secondmicrofluidic channel and the first array of islands are further arrangedto generate inertial lift forces that substantially prevent the group ofthe first type of particle from propagating with the siphoned fluidportion through the openings of the first array.

In another aspect, the subject matter of the present disclosure may beembodied in methods of shifting particles between fluid samples in amicrofluidic device, the methods including: flowing a first fluid samplecontaining multiple first types of particle into a particle sortingregion of the microfluidic device, in which the particle sorting regionincludes a first microfluidic channel, a second microfluidic channelextending along a first side of the first microfluidic channel, a firstarray of islands separating the first microfluidic channel from thesecond microfluidic channel, a third microfluidic channel extendingalong a second side of the first microfluidic channel, and a secondarray of islands separating the first microfluidic channel from thethird microfluidic channel, the second side being opposite to the firstside of the first microfluidic channel, each island in the first arraybeing separated from an adjacent island in the first array by an openingthat fluidly couples the first microfluidic channel to the secondmicrofluidic channel, and each island in the second array beingseparated from an adjacent island in the second array by an opening thatfluidly couples the first microfluidic channel to the third microfluidicchannel; and flowing a second fluid sample into the particle sortingregion, in which a fluidic resistance of the second microfluidic channelchanges relative to a fluidic resistance of the first microfluidicchannel along a longitudinal direction of the particle sorting regionsuch that a portion of the first fluid sample passes from the firstmicrofluidic channel into the second microfluidic channel throughopenings between islands in the first array, a fluidic resistance of thethird microfluidic channel changes relative to the fluidic resistance ofthe first microfluidic channel along the longitudinal direction of theparticle sorting region such that a portion of the second fluid samplepasses from the third microfluidic channel into the first microfluidicchannel through openings between islands in the second array, and thefirst microfluidic channel, the second microfluidic channel and thefirst array of islands are further arranged to generate inertial liftforces that substantially prevent the multiple first types of particlefrom propagating with the portion of the first fluid sample that passesthrough the openings of the first array.

Implementations of the methods may have one or more of the followingfeatures. For example, in some implementations, both the first fluidsample and the second fluid sample are delivered to the firstmicrofluidic channel. The second fluid sample may continuously flowthrough the first microfluidic channel without being substantiallysiphoned through the openings into the second microfluidic channel. Theinertial lift forces may shift the group of the first type of particlesacross fluid streamlines so that the group of the first type ofparticles is transferred to the second fluid sample.

In some implementations, the first fluid sample is delivered to thefirst microfluidic channel and the second fluid sample is delivered tothe third microfluidic channel.

In some implementations, the inertial lift forces shift the multiplefirst types of particle across fluid streamlines so that the multiplefirst types of particle are transferred from the first fluid sample tothe second fluid sample within the first microfluidic channel.

In some implementations, the first fluid sample includes multiple secondtypes of particle, in which the multiple second types of particlepropagate with the fluid portion of the first fluid sample that passesinto the second microfluidic channel. The first type of particle can belarger than the second type of particle.

In some implementations, the first fluid sample includes a differentfluid than the second fluid sample.

In some implementations, the amount of the first fluid sample thatpasses from the first microfluidic channel into the second microfluidicchannel is substantially the same as the amount of the second fluidsample that passes from the third microfluidic channel into the firstmicrofluidic channel so that a concentration of the first type ofparticle within the first microfluidic channel remains substantiallyconstant.

In some implementations, the change in the fluidic resistance of thesecond microfluidic channel relative to the fluidic resistance of thefirst microfluidic channel includes an increase in a cross-sectionalarea of the second microfluidic channel along the longitudinaldirection.

In some implementations, the change in the fluidic resistance of thethird microfluidic channel relative to the fluidic resistance of themicrofluidic channel includes an increase in a cross-sectional area ofthe third microfluidic channel along the longitudinal direction.

In some implementations, the first fluid sample includes multiple secondtypes of particle, and the multiple second types of particle propagatewith the fluid portion of the first fluid sample that passes into thesecond microfluidic channel. The first type of particle can be largerthan the second type of particle.

The first type of particle may have an average diameter between about 1μm and about 100 μm.

In another aspect, the subject matter of the present disclosure may beembodied in a method of sorting particles in a fluid sample, in whichthe method includes flowing a fluid sample containing a group of a firsttype of particle and a group of a second type of particle into aparticle sorting region of a microfluidic device, in which the particlesorting region includes a first microfluidic channel, a secondmicrofluidic channel extending along the first microfluidic channel, anda first array of islands separating the first microfluidic channel fromthe second microfluidic channel, in which a fluidic resistance of thefirst microfluidic channel relative to the fluidic resistance of thesecond microfluidic channel changes along a section of the particlesorting region such that a first portion of the fluid sample is siphonedfrom the first microfluidic channel into the second microfluidic channelthrough openings between islands in the first array, and in which thefirst microfluidic channel, the second microfluidic channel and thefirst array of islands are further arranged to generate inertial liftforces that substantially prevent the group of the first type ofparticle from propagating with the siphoned fluid portion through theopenings of the first array while allowing the group of the second typeof particle to propagate with the siphoned fluid portion into the secondmicrofluidic channel.

Implementations of the method may have one or more of the followingfeatures. For example, in some implementations, the inertial lift forcesshift the group of the first type of particles across fluid streamlinesso that the group of the first type of particles continue to propagatewith a second portion of the fluid sample remaining in the firstmicrofluidic channel. In some implementations, the first type ofparticle is larger than the second type of particle. In someimplementations, the first portion of the fluid sample passes throughthe openings in the first array of islands in response to a change inthe fluidic resistance between the first microfluidic channel and thesecond microfluidic channel. The change in the fluidic resistance mayinclude a change in a cross-sectional area of one of the firstmicrofluidic channel or the second microfluidic channel along adirection of fluid flow. The change in the fluidic resistance mayinclude a change in a size of the openings between the islands in thearray.

Implementations of the subject matter described herein provide severaladvantages. For example, in some implementations, the subject matterdescribed herein can be used to isolate particles within a fluid and/orfocus particles within a fluid. In some implementations, the subjectmatter described herein can be used to filter particles from a fluid orshift particles from one fluid to another fluid. High volumetriccapacity and throughput, substantial and tunable fluid volume reduction,and high particle yields with inexpensive and simple instruments can beachieved using the techniques described herein. In some implementations,the presently described techniques also may provide streamlinedprocessing and simple integration with other microfluidic modules. Forclinical applications, the systems described herein may be configured asboth self-contained and disposable. In contrast, forbioprocessing/industrial applications, the devices may be configured forcontinuous flow/processing.

For the purposes of this disclosure, channel refers to a structure inwhich a fluid may flow.

For the purposes of this disclosure, microfluidic refers to a fluidicsystem, device, channel, or chamber that generally have at least onecross-sectional dimension in the range of about 10 nm to about 10 mm.

For the purposes of this disclosure, gap refers to an area in whichfluids or particles may flow. For example, a gap may be a space betweentwo obstacles in which fluids flow.

For the purposes of this disclosure, rigid island structure refers to aphysical structure through which a particle generally cannot penetrate.

For the purposes of this disclosure, fluidic resistance refers to theratio of pressure drop across a channel (e.g., a microfluidic channel)to the flow rate of fluid through the channel.

Particles within a sample can have any size which allows them totransported within the microfluidic channel. For example, particles canhave an average hydrodynamic size that is between 1 μm and 100 μm. Theparticle size is limited only by channel geometry; accordingly,particles that are larger and smaller than the above-described particlescan be used. The size of particles (e.g., cells, eggs, bacteria, fungi,virus, algae, any prokaryotic or eukaryotic cells, organelles, exosomes,droplets, bubbles, pollutants, precipitates, organic and inorganicparticles, magnetic beads, and/or magnetically labeled analytes), suchas the average hydrodynamic particle size or average diameter, can bedetermined using standard techniques well known in the field.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods, materials,and devices similar or equivalent to those described herein can be usedin the practice or testing of the present invention, suitable methods,materials and devices are described below. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

The details of one or more embodiments are set forth in the accompanyingdrawings and the description below. Other features, objects, and will beapparent from the description, drawings, and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic that illustrates a top view of an example of amicrofluidic device capable of shifting the position of particles withinand across fluid streamlines.

FIG. 2 is a schematic that illustrates a top view of an example of adevice for particle and fluid shifting, in which a fluid is allowed tocross over a channel through which particles are propagating.

FIG. 3 is a schematic that illustrates a top view of an example of adevice having two inlet microfluidic channels coupled to a mergingchannel, which, in turn, is coupled to a particle shifting area.

FIG. 4 is a schematic that illustrates a top view of an example of adevice having a particle shifting area for size-based sorting ofparticles.

FIG. 5 is a schematic that illustrates a top view of an example of adevice having a particle shifting area for size-based sorting ofparticles.

FIG. 6A is a schematic that illustrates a top view of an example of amicrofluidic system that includes a particle sorting area.

FIG. 6B is an enlarged view of the particle sorting area of FIG. 6A.

FIGS. 7 and 8 are schematics illustrating top views of example particlesorting regions.

FIG. 9 is a series of plots (FIGS. 9A-9D) illustrating debulkingperformance for a device that performs fractionation based on inertiallift forces.

FIG. 10 is a series of plots (FIGS. 10A-10B) of white blood cell (WBC)yield against fluid flow rate.

FIG. 11 is a plot of yield of different sized fluorescent beads acrossdifferent flow rates.

FIG. 12 is a plot of WBC yield versus fluid shift.

FIG. 13 is a photograph of a microscope slide that accommodates amultiplexed array of fluid shifting devices.

DETAILED DESCRIPTION

Interactions between particles (e.g., cells, e.g., blood cells ingeneral as well as fetal blood cells in maternal blood, bone marrowcells, and circulating tumor cells (CTCs), sperm, eggs, bacteria, fungi,virus, algae, any prokaryotic or eukaryotic cells, cell clusters,organelles, exosomes, droplets, bubbles, pollutants, precipitates,organic and inorganic particles, beads, bead labeled analytes, magneticbeads, and/or magnetically labeled analytes), the fluids in which theytravel (e.g., blood, aqueous solutions, oils, or gases), and rigidstructures can be used to shift particles across fluid streamlines inmicrofluidic devices in a controlled fashion. In particular, forcesexperienced by particles traveling in a microfluidic device can be usedto precisely position the particles, such that a variety of usefulmicrofluidic operations can be performed. Examples of microfluidicoperations that can be performed using such forces include, but are notlimited to, concentrating particles in a carrier fluid, shiftingparticles from one carrier fluid to another fluid, separating particleswithin a fluid based on particle size (e.g., average diameter), focusingparticles within a carrier fluid to a single-streamline (or to multipledifferent streamlines), precise positioning of particles at any positionwithin a micro-channel, and mixing (defocusing) particles. Moreover, anyof the above operations can be executed simultaneously with othertechniques (e.g., magnetic sorting) to enhance the operation'seffectiveness.

Several different mechanisms can be employed to create the forcescapable of shifting particles across fluid streamlines. A first type offorce is referred to as “bumping” (also called deterministic lateraldisplacement (DLD)). Bumping is direct interaction between a rigid wallof a structure and a particle that arises due to the size of theparticle relative to the wall. Since the center of a particle havingradius r_(p) cannot pass closer to an adjacent structure than r_(p), ifthe particle center lies on a streamline that is less than r_(p) fromthe structure, the particle will be bumped out by the structure to adistance that is at least r_(p) away. This bumping may move the particleacross fluid streamlines.

Another type of force is called inertial lift force (also known as wallforce or wall induced inertia). In contrast to bumping, the inertiallift force is a fluidic force on a particle, not a force due to contactwith a rigid structure. Though not well understood, the inertial liftforce is a repulsive force arising due to a flow disturbance generatedby the particle when the particle nears the wall. A particle flowingnear a micro-channel wall experiences an inertial lift force normal tothe wall. At high flow rates, the inertial lift force is very strong andcan shift the particle across streamlines. Furthermore, because theforce is highly size-dependent (larger particles experience a muchlarger force), it can be employed to fractionate particles based onsize. Further details on inertial flow can be found in D. Di Carlo, D.Irimia, R. G. Tompkins, and M. Toner, “Continuous inertial focusing,ordering, and separation of particles in microchannels.,” Proc. Natl.Acad. Sci. U.S.A., vol. 104, no. 48, pp. 18892-18897, November 2007; D.Di Carlo, J. F. Edd, K. J. Humphry, H. A. Stone, and M. Toner, “Particlesegregation and dynamics in confined flows.,” Phys. Rev. Lett., vol.102, no. 9, p. 094503, March 2009; and D. Di Carlo, “Inertialmicrofluidics,” Lab Chip, vol. 9, no. 21, p. 3038, 2009, each of whichis incorporated herein in its entirety.

Another type of force is a result of pressure drag from Dean flow.Microfluidic channels having curvature can create additional drag forceson particles. When introducing the curvature into rectangular channels,secondary flows (i.e., Dean flow) may develop perpendicular to thedirection of a flowing stream due to the non-uniform inertia of thefluid. As a result, faster moving fluid elements within the center of acurving channel can develop a larger inertia than elements near thechannel edges. With high Dean flow, drag on suspended particles withinthe fluid can become significant.

Another type of force occurs with High Stokes number flow. The Stokesnumber (Stk) describes how quickly a particle trajectory changes inresponse to a change in fluid trajectory. For Stk greater than 1, a lagexists between the change in fluid trajectory and the change in particletrajectory. Under high Stokes flow conditions (e.g., a Stokes numbergreater than about 0.01), changing the fluid flow direction can be usedto force particles across streamlines. Further details on Dean flow andhigh Stokes number can be found, for example, in U.S. Pat. No.8,186,913, which is incorporated herein by reference in its entirety. Inboth high Stokes flow applications and Dean flow applications, the fluiddisplacement causes the particles to cross fluid streamlines.

Other techniques for shifting particles include viscoelastic andinertio-elastic focusing. Details on those methods can be found in“Sheathless elasto-inertial particle focusing and continuous separationin a straight rectangular microchannel,” Yang et al., Lab Chip (11),266-273, 2011, “Single line particle focusing induced by viscoelasticityof the suspending liquid: theory, experiments and simulations to designa micropipe flow-focuser,” D'Avino et al., Lab Chip (12), 1638-1645,2012, and “Inertio-elastic focusing of bioparticles in microchannels athigh throughput,” Lim et al., Nature Communications, 5 (5120), 1-9,2014, each of which is incorporated herein by reference in its entirety.

The foregoing techniques are “internal,” in that they use fluid flowand/or structures of the microfluidic channel itself to generate theforces necessary to shift particles across streamlines. In some cases,other external mechanisms can also be used in conjunction with one ormore of the internal forces to alter the course of particles travelingwithin a fluid. For example, in some cases, externally applied magneticforces, gravitational/centrifugal forces, electric forces, or acousticforces may be used to cause a shift in particle position across fluidstreamlines. Further information on how to apply such forces can befound, e.g., in WO 2014/004577 titled “Sorting particles using highgradient magnetic fields,” U.S. Pat. No. 7,837,040 titled “Acousticfocusing,” WO 2004/074814 titled “Dielectrophoretic focusing,” and“Microfluidic, Label-Free Enrichment of Prostate Cancer Cells in BloodBased on Acoustophoresis,” Augustsson et al., Anal. Chem. 84(18), Sep.18, 2012.

The present disclosure focuses primarily on combining inertial liftforces with periodic fluid extraction to shift particles across fluidstreamlines for sorting particles between fluids and/or separatingparticles from fluids. In particular, a particle containing first fluidis introduced into a first microfluidic channel having arrays of rigidisland structures separating the first channel from adjacentmicrofluidic channels. As the first fluid is extracted from the firstmicrofluidic channel into the second microfluidic channel through gapsbetween the island structures, the particles are drawn nearer to theisland structures. As the particles reach nearer to the islandstructures, the particles experience an inertial lift force away fromthe direction of fluid extraction such that the particles cross fluidstreamlines and remain within the first channel. At the same time, asecond fluid passes from a third microfluidic channel through a secondarray of island structures into the first microfluidic channel, suchthat the particles in the first channel are transferred to the secondfluid within the first channel. In some implementations, the amount ofthe first fluid entering the second channel from the first channel isthe same as the amount of the second fluid entering the first channelfrom the third channel. As a result, a particle may be shifted from onefluid to another fluid while maintaining the same particleconcentration. The combination of fluid extraction and inertial liftforces may be used to perform positioning of particles, particlefiltering, particle mixing, fluid mixing, and/or shifting of fluidsacross particle streams, among other operations.

It should be noted, however, that the techniques described herein forparticle and/or fluid sorting are not limited to using inertial liftforces. Instead, periodic fluid extraction also may be combined with oneor more of the above-described forces (both internal and external) tocontrol the position of particles within fluids propagating in amicrofluidic device.

The mechanisms for shifting particles disclosed herein may also besize-based and therefore can be used to perform size-based manipulationof particles (e.g., based on the average diameter of the particles).Through repeated shifting of particles across streamlines, both fluidand particles in microfluidic devices can be manipulated to performoperations such as focusing particles to one or more fluid streamlines,filtering particles from a fluid, mixing different particles fromdifferent fluid streams, and/or sorting particles based on size. Ingeneral, “focusing” particles refers to re-positioning the particlesacross a lateral extent of the channel and within a width that is lessthan the channel width. For example, the techniques disclosed herein canlocalize particles suspended in a fluid to a fluid stream, in which theratio of the channel width to the width of the fluid stream is about1.05, 2, 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100. Particlesmay have various sizes including, but not limited to, between about 1 μmand about 100 μm in average diameter.

The mechanisms for shifting particles disclosed herein may also dependon particle shape (e.g., spherical vs. cylindrical) and deformability(e.g., rigid vs. compliant), thereby enabling differential manipulationand sorting of particles based on shape and deformability.

Particle Shifting/Sorting Using Inertial Lift Forces

Prior to discussing how particles may be shifted from one fluid toanother using one or more of the devices disclosed herein, it is helpfulto first review fluid extraction and inertial forces within the contextof a more basic device structure, such as the device shown in FIG. 1.FIG. 1 is a schematic that illustrates a top view of an example of amicrofluidic device 100 capable of shifting/sorting the position ofparticles 102 across fluid streamlines while the fluid propagatesthrough the microfluidic device 100. As will be explained, the particleshifting across fluid streamlines relies on the inertial lift forcesexperienced by particles as fluid is periodically extracted from amicrofluidic channel. For reference, a Cartesian coordinate system isshown, in which the x-direction extends into and out of the page.

During operation of the device 100, a fluid carrying the particles 102is introduced through an inlet microfluidic channel 104. In this andother implementations of the particle shifting devices, the fluid can beintroduced through the use of a pump or other fluid actuation mechanism.The inlet channel 104 splits into a particle sorting region having twodifferent fluid flow channels (second microfluidic channel 106 and firstmicrofluidic channel 108 substantially parallel to the secondmicrofluidic channel 106) that are separated by a 1-dimensional array ofrigid island structures 110. The 1-dimensional array of islandstructures 110 extends substantially in the same direction as the flowof the fluid through the second and first microfluidic channels. Eachisland structure 110 in the array is separated from an adjacent island110 by an opening or gap 114 through which fluid can flow. Each gap 114in the example of FIG. 1 has the same distance between adjacent islands110. In other implementations, different gaps can have differentdistances between adjacent islands 110. For example, in someimplementations, a length of each subsequent opening (e.g., as measuredalong the fluid propagation direction—the z-direction in FIG. 1) in thefirst array is greater than a size of a previous opening in the array.Alternatively, in some implementations, the distance can alternatebetween larger and smaller for subsequent openings. Furthermore,although a 1-dimensional array is shown in FIG. 1, the islands 110 maybe arranged in different configurations including, for example, atwo-dimensional array of islands. The boundaries of the fluid flowregions within the microfluidic channels are defined by the device walls112 and the walls of the islands 110.

As the fluid propagates substantially along the z-direction from theinlet channel 104 to the channels (106, 108), particles 102 experience aforce (in this example, an inertial lift force) that causes theparticles 102 to shift across fluid streamlines and travel along thefirst microfluidic channel 108. These inertial lift forces are in thenegative y-direction (see short arrows adjacent to each particle 102 inFIG. 1).

For instance, when a particle 102 is located in the inlet channel 104and approaches the top wall 112, the particle experiences an inertiallift force that pushes the particle down toward the first microfluidicchannel 108. Once in the first microfluidic channel 108, the particle102 may approach a wall of the first island 110, such that it againexperiences an inertial lift force pushing the particle 102 down,maintaining the particle within the first microfluidic channel 108. Therepeated application of the inertial lift force to the particle 102 ineach of the “particle shift” regions shown in FIG. 1 thus serves toseparate/filter the particle from the fluid propagating through thesecond microfluidic channel 106.

At the same time, portions of the fluid traveling in the firstmicrofluidic channel 108 are extracted or flow into the secondmicrofluidic channel at one or more “fluid shift” or “fluid extraction”regions (see FIG. 1) in the device 100. In the example of FIG. 1, eachfluid shift region corresponds to an opening or gap that extends betweenthe first microfluidic channel 108 and the second microfluidic channel106. Each “fluid shift” region primarily allows fluid to be extractedfrom the first microfluidic channel 108 into the second microfluidicchannel 106. The movement of fluid into the gaps tends to pull theparticles 102 toward the gaps as well, since the particles follow thefluid streamlines. However, as the particles move closer to the gaps114, they approach the island structures 112, which impart an inertiallift force causing the incident particles to cross fluid streamlines ina direction away from the gaps 114. That is, the particles 102 shiftfrom a fluid streamline passing into the second microfluidic channel 106to a fluid streamline that continues to flow in the first microfluidicchannel 108. As a result, the particles 102 continue to propagate in thefirst microfluidic channel 108 and are not shifted into the secondmicrofluidic channel 106 with the fluid. If there were no fluid shiftingfrom the first microfluidic channel 108 to the second microfluidicchannel 106, the particles would migrate as a result of inertialfocusing. However, by shifting the fluid across the channels, theparticles 102 tend to follow the fluid toward areas where the inertiallift force is much stronger than the shear gradient force, thus causingthe particles to shift across streamlines in a very efficient andcontrolled manner.

In the present example, the fluid is extracted through the fluid shiftregions as a result of decrease in fluidic resistance. That is, for afluid of constant viscosity, the gaps 114 between adjacent islands 110increase the channel area through which the fluid can flow, resulting ina reduced fluidic resistance. As fluid propagates through channel 108 ofdevice 100 and arrives at a gap 114, a portion of the fluid will flowinto the gap 114 and subsequently into the second microfluidic channel106 (i.e., the fluid portion is extracted into channel 106). Thedecrease in fluidic resistance also can occur as a result of theincreasing channel width in the second microfluidic channel 106. In someimplementations, the width of channel 106 may be understood as thedistance between a position on the second channel wall 112 and aposition on a surface of an island 110 directly across from and facingthe position on the second channel wall 112. For the gap regions betweenislands 110, the width of the channel 106 may be understood, in someimplementations, as the distance between a position on the secondchannel wall 112 and a position on an imaginary surface extendingthrough the gap between adjacent islands 110 that is directly acrossfrom and facing the position on the channel wall 112, in which theimaginary surface is co-linear with the sides of the adjacent islandsclosest to and facing the wall 112.

In a particular example, such as shown in FIG. 1, the secondmicrofluidic channel wall 112 is slanted at an angle away from (orientedobliquely with respect to) the islands so that the width of the secondmicrofluidic channel 106 increases along the channel's longitudinaldirection (e.g., in the direction of fluid propagation or the positivez-direction for the example shown in FIG. 1), thus causing a decrease influidic resistance. Any increase in the cross-sectional area of thechannel 106 along the longitudinal direction of the first microfluidicchannel, not just an increase in width, also can be employed to reducethe fluidic resistance. Alternatively, or in addition, the fluid mayexperience an increase in fluidic resistance in channel 108 relative tothe fluidic resistance of channel 106 (e.g., through a decrease in thecross-sectional area of the channel 108 along the longitudinaldirection).Thus, it may be said that the fluid is extracted in responseto a change in the relative fluidic resistance between the second andfirst microfluidic channels that leads to fluid being extracted from thefirst channel 108 into the second channel 106. The change in therelative fluidic resistance may occur over the entire particle sortingregion or over a portion of the sorting region that is less than theentire particle sorting region. The change in the relative fluidicresistance may occur along the direction of the fluid flow through theparticle sorting region (e.g., along a longitudinal direction of theparticle sorting region as shown in FIG. 1).

With progressively lower fluidic resistance at the gaps 114 and/or inchannel 106, greater amounts of fluid flow into the second microfluidicchannel 106. Furthermore, the repeated shifting of fluid into the secondchannel 106 reduces the amount of fluid in the first channel 108. Forthe configuration shown in FIG. 1, the repeated fluid extraction thusincreases the particle-to-fluid concentration in the first channel 108,while decreasing the concentration of particles in the secondmicrofluidic channel 106, such that the fluid in the second microfluidicchannel 106 is “filtered” or “purified.”

The resulting focused particle streamline may be coupled to a separateprocessing region of the microfluidic device 100 or removed from thedevice 100 for additional processing and/or analysis. Likewise, the“filtered” fluid in the second channel 106 may be coupled to a separateregion of the microfluidic device 100 or removed from the device 100 foradditional processing and/or analysis. In some implementations, theparticles 102 entering the device 100 are “pre-focused” to a desiredfluid streamline position that is aligned with the first microfluidicchannel 108. By pre-focusing the particles 102 to a desired position,the probability that particles inadvertently enter into the secondmicrofluidic channel 106 can be reduced.

Another advantage of the particle shifting techniques described hereinis that it may be used to focus the particles along one or morestreamlines. For instance, as previously explained, portions of fluidmay be extracted from an initial microfluidic channel into one or moreparallel microfluidic channels. In some instances, the parallelmicrofluidic channels containing the extracted fluid then may bere-combined with the initial microfluidic channel downstream so that theparticles are confined to designated streamlines in a single channel. Anadvantage of this technique of combining fluid shifting with inertiallift force is that particles may be focused to desired positions withinthe downstream channel (e.g., near the channel wall, at the middle ofthe channel, or halfway between the channel wall and the middle of thechannel, among other positions) by controlling how much fluid is removedfrom each side of the initial channel, providing increased flexibilityto the design and use of microfluidic devices. In contrast, formicrofluidic systems based primarily on inertial focusing, one haslimited ability to choose the position of the focused stream within thechannel.

Having provided a review of fluid extraction and inertial forces,microfluidic devices for shifting/sorting particles between fluids cannow be described. In particular, the fluid and particle shiftingtechniques described herein can be used to shift fluids across channels,without an accompanying shift in particles such that the particles areindirectly transferred to another fluid. FIG. 2 is a schematic thatillustrates an example of a device 200 for shifting particles betweenfluids. The device 200 includes a first inlet microfluidic channel 204and a second inlet microfluidic channel separated by a dividingstructure 205, such as a wall or other object that prevents mixingbetween the first inlet 204 and the second inlet 206. At the end of thedividing structure 205, the first and second inlet microfluidic channels(204, 206) are fluidly coupled to a particle shifting area that hasthree different fluid flow regions (a second microfluidic channel 208, afirst microfluidic channel 210, and a third microfluidic channel 212).

The second microfluidic channel 208 is separated from the firstmicrofluidic channel 210 by a first array 214 of island structures 218.The third microfluidic channel 212 is separated from the firstmicrofluidic channel 210 by a second array 216 of islands 218. Eachadjacent island structure in the first array 214 and each adjacentisland structure in the second array 216 is separated by a gap for fluidshifting. The boundaries of the microfluidic channels are defined by thedevice walls 220 and the walls of the islands. The microfluidic channelwall 220 of the second channel 208 is slanted at an angle away from(oriented obliquely with respect to) the islands 218 so that the widthof the second channel increases along the fluid propagation direction,thus causing a decrease in fluidic resistance and leading to extractionof fluid from the first channel 210 into the second channel 208. Incontrast, the wall 220 of the third channel 212 is slanted at an angletoward the islands 218 so that the width of the third channel 212decreases along the fluid propagation direction, thus causing anincrease in fluidic resistance, and leading to fluid being extractedfrom the third channel 212 into the first channel 210.

During operation of the device 200, particles 202 flowing within a firstfluid in the first inlet channel 204 interact with the channel wallssuch that they experience inertial lift forces, which shift or focus theparticles 202 toward the center streamlines of the fluid flow. The fluidpathway of the central microfluidic channel 210 is substantially alignedwith the fluid pathway of the first inlet channel 204 so the focusedparticles from channel 204 and a portion of the first fluid flow intothe first channel 210. Once the particles 202 enter the firstmicrofluidic channel 210, they experience inertial lift forces from theisland structures 218 that continue to focus the particles 202 along oneor more central streamlines extending through the channel 210. At thesame time, some of the first fluid is extracted into the secondmicrofluidic channel 208 in the “fluid shift” regions due to the reducedfluidic resistance. Because the particles 202 experience inertial liftforces in the first channel 210, the majority of particles 202 remain inthe first channel 210 and are not carried with the first fluid into thesecond channel 208.

A second fluid is provided in the second inlet channel 206. The secondfluid may be the same fluid as the carrier fluid used to introduce theparticles from inlet channel 204 or a different fluid. The second fluidprimarily flows from second inlet 206 into the third microfluidicchannel 212. Portions of the second fluid are extracted from inlet 206and/or third channel 212 into the first microfluidic channel 210 in the“fluid shift” regions. The extraction of the second fluid occurs as aresult of the increasing fluidic resistance (e.g., decreasing channelwidth) the second fluid experiences as the second fluid propagates downchannel 212. Accordingly, an increasing amount of the second fluidbegins to flow within the first channel 210. In some implementations,e.g., with long enough microfluidic channels, the second fluid may evencross-over from the third microfluidic channel 212 into the firstmicrofluidic channel 210 and finally into the second microfluidicchannel 208 to combine with the first fluid. However, because theparticles 202 experience the inertial lift forces in the first channel210, the majority of particles 202 remains in the first channel 210 andare not carried along with the first or the second fluid into the secondchannel 208. As a result, the combination of the fluid shift regions andthe particle shift regions allows isolation of particles 202 from thefirst fluid. That is, the particles 202 are shifted from the first fluidinto the second fluid as the second fluid is introduced into channel210. Since inertial forces maintain the particles 202 within channel210, the particles also may be isolated, in certain implementations,from the merged first and second fluids traveling through channel 208.

If the amount of the first fluid extracted from the first channel 210into the second channel 208 is kept equal to or substantially equal tothe amount of the second fluid introduced to the first channel from thethird channel over the length of the device, then the amount of fluidpropagating through channel 210 may be kept substantially constant.Similarly, since inertial lift forces cause the number of particlespropagating through channel 210 to remain substantially constant, thetotal concentration of particles does not appreciably change even as thefluid changes within channel 210. That is, the concentration ofparticles 202 within the first fluid at the beginning of the channel 210is substantially the same as the concentration of particles 202 withinthe second fluid near the end of the channel 210, after the fluid shifthas been completed.

In some implementations, the first fluid is not entirely extracted fromchannel 210 into channel 208. Rather, the microfluidic device may beconfigured so that, after operation of the device, there is acombination of both the first fluid and the second fluid within channel210. In such cases, the first fluid and second fluid may propagate sideby side in accordance with laminar flow or may be mixed as a result of,e.g., diffusion. In either instance, if the amount of the first fluidextracted into channel 208 is substantially the same as the amount ofsecond fluid introduced into channel 210 from channel 212 over thelength of the device, then the concentration of particles 202 relativeto whatever fluids are within channel 210 may be kept substantiallyconstant.

Any of the fluid streams from the second, first, or third channels maybe coupled to a separate region of the microfluidic device or removedfrom the device for additional processing or analysis. In someimplementations, the variation in size/fluidic resistance of the secondand third channels can be set so as to ensure that equal amounts offluid flow in from the third channel and out the second channel at eachunit.

Another example of a device capable of causing particles to transitionbetween fluids is shown in FIG. 3, which is a schematic that illustratesan example of a device 300 that includes two inlet microfluidic channels(304, 306) coupled to a single microfluidic channel 305 for merging thefluids. The merging channel 305 is, in turn, coupled to a particleshifting area that includes two different flow regions (secondmicrofluidic channel 308 and first microfluidic channel 310). The secondmicrofluidic channel 308 is separated from the first microfluidicchannel 310 by an array of island structures 312, in which each island312 is separated from an adjacent island 312 by a gap 314 for fluidshifting. In addition, the top wall 316 of the second microfluidicchannel 308 is slanted at an angle away from the islands 312 in order todecrease the fluidic resistance along the z-direction.

During operation of the device 300, a first fluid (“Fluid 1”) containingparticles 302 is introduced in the first inlet channel 304 and a secondfluid (“Fluid 2”) having no particles is introduced into the secondinlet channel 306. Assuming the fluids are introduced at flow ratescorresponding to low Reynolds numbers (and thus laminar flow), there islittle mixing between the two different fluids in the merge region 305,i.e., the two fluids essentially continue flowing as layers adjacent toone another. The fluid pathway within the merge region 305 is alignedwith the fluid pathway of the first microfluidic channel 310 such thatthe merged fluids primarily flow into the first channel 310. As the twofluids enter the first microfluidic channel 310, the particles 302within the first fluid experience inertial lift forces from the islandstructures 312 that are transverse to the direction of flow and thatkeep the particles 302 within the first microfluidic channel.

At the same time, the increasing width of the second microfluidicchannel 308 (due to the slanted channel wall 316) decreases the fluidicresistance, such that portions of the first fluid (which is nearest tothe island structures) are extracted into the second channel 308 at eachgap between the islands 312. Because the first fluid flows as a layerabove the second fluid, little to none of the second fluid is extractedinto the second channel 308. After propagating for a sufficient distancepast the islands 312, most of the first fluid is extracted into thesecond channel 308, whereas the particles 302 and most or all of thesecond fluid remain in the first channel 310. Accordingly, themicrofluidic device configuration shown in FIG. 3 is also useful fortransferring particles from one fluid to a second different fluid. Ifthe amount of the first fluid flowing through inlet 304 is substantiallythe same as the amount of the second fluid flowing through inlet 306,then the concentration of particles 302 in the second fluid withinchannel 310 (and after extraction of the first fluid) can be keptsubstantially the same as the concentration of particles 302 in thefirst fluid within the inlet 304. In some implementations, thepropagation distance is long enough so that the second fluid also isextracted into the second microfluidic channel 308. In that case, theconcentration of the particles 302 in the second fluid within firstmicrofluidic channel 310 can be increased to a level that is higher thanthe particle concentration within channel 304.

In some implementations, repeated particle and fluid shifting can beused to perform size-based separation of particles within a fluid. FIG.4 is a schematic that illustrates an example of a device 400 being usedfor size-based sorting of particles. The device configuration isidentical to the device 300 shown in FIG. 3. During operation of thedevice 400, a first fluid (“Fluid 1”) containing particles of differentsizes (large particles 402 and small particles 403) is introduced intoin the first inlet channel 404, and a second fluid (“Fluid 2”) having noparticles is introduced into the second inlet channel 406. The first andsecond fluids may be the same type or different types of fluids. Again,assuming the fluids are introduced at flow rates corresponding to lowReynolds numbers (and thus laminar flow), there is little mixing betweenthe two different fluids in the merge region 405, i.e., the two fluidsessentially continue flowing as layers adjacent to one another. As thetwo fluids enter the first microfluidic channel 410, the forces on thelarger particles 402 are great enough to keep the particles 402 withinthe first microfluidic channel 410. In contrast, the forces on thesmaller particles 403 are not high enough to prevent the small particles403 from being extracted with the first fluid into the secondmicrofluidic channel 408. After repeated particle shifting and fluidextraction over a sufficient distance, most of the first fluid and thesmall particles 403 are extracted into the second channel 408, whereasthe large particles 402 and most of the second fluid remain in the firstchannel 410. This process, also called fractionation, is useful forseparating particles from a fluid based on size.

There are multiple reasons why large particles 402 are preferentiallyretained over the smaller particles 403. First, the inertial lift forceis highly nonlinear in particle diameter. For instance, it is believedthat near channel walls, the inertial lift force scales in the range ofa³ to a⁶ where a is the particle diameter, such that large particlesexperience a much larger force than small particles. The larger inertiallift force may be used to move particles out of the fluid streamsadjacent to the islands that shift upward from one from one row of thearray of island structures to the next. Further information on therelation between particle size and the inertial lift force may be foundin Di Carlo et al., “Particle Segregation and Dynamics in ConfinedFlows”, Physical Review Letters, 2009, incorporated herein by referencein its entirety. Second, the equilibrium position of large particles isgenerally farther from the wall than that of small particles, andtherefore is further from the fluid extraction channel and more likelyto lie on a streamline that does not shift toward the extractionchannel. The large particles therefore may be retained within a givenrow, whereas smaller particles flowing near the island shift upward fromone row of the array to next.

Thus, fractionation is accomplished by repeatedly (1) using the inertiallift force to move large particles away from a channel wall and then (2)shifting the fluid that is free of large particles into an adjacentchannel. In some implementations, fractionation can also be used to sortparticles from a source fluid (e.g., blood) across fluid streamlinesinto an adjacent destination fluid (e.g., buffer).

For instance, FIG. 5 is a schematic illustrating an example of a device500 that also can be used for separating particles based on size. Theconfiguration of the device 500 is the same as the device 200 shown inFIG. 2. The fluidic resistance in the third microfluidic channel 512progressively increases due to decreasing channel width, whereas thefluidic resistance of the second microfluidic channel 508 progressivelydecreases due to increasing channel width. Accordingly, during operationof the device 500, repeated fluid shifting of a first fluid (“Fluid 1”)from the first microfluidic channel 510 into the second microfluidicchannel 508 occurs at the gaps between islands 518 in the first array514. Similarly, repeated fluid shifting of a second fluid (“Fluid 2”)from the third microfluidic channel 512 into the first microfluidicchannel 510 occurs at the gaps between islands 518 in the second array516. The fluid extraction forces are large enough to pull the smallparticles 503 along with the first fluid, but not great enough tocounter the inertial lift forces experienced by the large particles 502.As a result, the large particles remain flowing along streamlines withinthe first microfluidic channel 510. After repeated particle and fluidshifting, the large particles 502 begin flowing along streamlines withinthe second fluid that has been shifted into the first channel 510. Ifthe amount of fluid flowing out of channel 510 into channel 508 is keptsubstantially equal to the amount of fluid flowing out of channel 512into channel 510 over the length of the particle sorting/shiftingregion, then the amount of fluid flowing within channel 510 can be keptsubstantially constant.

The microfluidic devices shown in FIGS. 1-5 implement particle shiftingacross fluid streamlines using inertial lift forces from themicrofluidic channel walls and from the periodic arrays of islandstructures. Techniques other than inertial lift force may be used toassist the shift of particles across fluid streamlines. For example,internal forces arising due to high Dean flow and/or high Stokes flow,such as inertial focusing, can be used to shift particles across fluidstreamlines and/or to maintain particles within a microfluidic channel.Alternatively, or in addition, external forces such as magnetic forces,acoustic forces, gravitational/centrifugal forces, optical forces,and/or electrical forces may be used to shift particles across fluidstreamlines. Additionally, the shape of the rigid island structures thatseparate different flow regions is not limited to the shapes shown inFIGS. 1-5. For example, the rigid island structures may have shapessimilar to posts, cuboids, or other polyhedrons in which the top andbottom faces are, or can be, congruent polygons. In some circumstances,such as at high flow rates, it is advantageous to use islands withstreamlined, tapered ends, as this helps minimize the formation of flowrecirculations (eddies) that disrupt flow in unpredictable andundesirable ways. Other shapes for the rigid island structures are alsopossible. The long axis of the rigid island structures may be orientedat an angle with respect to the average flow direction of the fluid, theaverage flow direction of the particles, or the long axis of the sortingregion. The shapes of the channel segments are not limited to theapproximately rectangular shapes shown in FIGS. 1-5. The channelsegments may include curves or substantial changes in width. Incross-section, the channels described in FIGS. 1-5 may be square,rectangular, trapezoidal, or rounded. Other shapes for the channelcross-sections are also possible. The channel depth may be uniformacross the particle sorting region, or the channel depth may varylaterally or longitudinally. Additionally, though FIGS. 1-5 show themicrofluidic channels as approximately rectilinear pathways, thechannels may be configured in other different arrangements. For example,in some implementations, the microfluidic channels may be formed to havea spiral configuration. For instance, the first microfluidic channel andthe second microfluidic channel may be arranged in a spiralconfiguration, in which the first and second microfluidic channel arestill be separated by the array of islands structures, but where thelongitudinal direction of fluid flow through the channels would follow agenerally spiral pathway. In some implementations, the dimensions orshape of the island structures may vary along the length of the sortingregions (e.g., in the direction of fluid flow) and/or along the width ofthe sorting regions (e.g., transverse to the direction of fluid flow).In some implementations, the percentage of fluid passing between islandstructures varies for different locations within the channel. Forexample, the percentage of fluid may be higher or lower through a firstgap between two island structures than the percentage of fluid passingthrough a next adjacent gap between two island structures.

Although some implementations shown in FIGS. 1-5 include two inletchannels, additional inlet channels may be coupled to the microfluidicchannels. In some implementations, three, four or more inlet channelsmay introduce fluid into the device regions that shift the particlesthrough fluid exchange and inertial lift forces. For example, in someimplementations, there may be three inlet channels, one which deliversblood, one which delivers staining reagents, and one which delivers abuffer stream. Using a combination of fluid shifting and inertial liftforce techniques disclosed herein, white blood cells from the bloodstream could be shifted into the reagent stream and then into a bufferstream.

In some implementations, the devices described herein may be used inconjunction with other microfluidic modules for manipulating fluidsand/or particles including, for example, filters for filteringsub-populations of particles of certain sizes. In addition, the devicesdescribed herein may be used in series and/or in parallel within amicrofluidic system.

Microfluidic Device Design Parameters

The effect of various design parameters on the operation of themicrofluidic device will now be described. For reference, FIG. 7 is aschematic illustrating a top view of an example particle sorting region700 containing several rows of island structures 710, with each row ofislands being separated from an adjacent row of islands by acorresponding interior microfluidic channel 703. Additionally, there isan exterior microfluidic channel 705 a extending above the array ofislands and an exterior microfluidic channel 705 b extending below thearray of islands. The primary direction of fluid flow is indicated bythe arrow 701. The width of the exterior channel 705 a (defined alongthe y-direction) expands along the length of the channel, whereas thewidth of the exterior channel 705 b (defined along the y-direction)contracts along the length of the channel. For the purposes of thefollowing discussion, the channels and islands may be understood asbeing arranged into separate “units” (see Unit 1, Unit 2 and Unit 3 inFIG. 7). Specifically, FIG. 7 illustrates three units of an array withtwo interior channels and two exterior channels.

The relevant design parameters for the particle sorting region 700 arethe unit length, width, and fluid shift. Here, the width, w, refers tothe dimension of the interior microfluidic channels 703, whereas thelength, l, refers to the length of an island structure 710 within aunit. The interior channels thus have fixed width w and fluidicconductance g. The expanding channel has widths w_(e,i) and fluidicconductances g_(e,i), where i refers to the unit number. Similarly, thecontracting channel has widths w_(e,i) and fluidic conductances g_(c,i),where i refers to the unit number. The fluid shift, f, is the fractionof the flow, q, in the interior channels that shifts between rows(channels) at each unit. The net flow in the interior channels does notchange because at each unit a flow fq (the product of f and q) shiftsout of these channels (at the openings 707 between island structures)and a flow fq shifts into these channels (at the openings 707 betweenisland structures). In contrast, the net flow in the exterior channels705 does change. The net flow in the contracting channel 705 b decreasesby fq at each unit, and the net flow in the expanding channel 705 aincreases by fq at each unit. Thus, the widths of the exterior channelsof each unit may be set to provide the desired shifting of fluid acrossthe array.

To each successive unit, fg (the product of f and g) is added to thefluidic conductance of the expanding channel and fg is subtracted fromthe fluidic conductance of the contracting channel. The changing fluidicconductance translates into a proportionate change in volumetric flowrate because the pressure drop per unit, p, is approximately the sameacross all channels in a unit and flow rate is related to fluidicconductance by the relation q=pg. Therefore, when the conductance of anexpanding channel (e.g., channel 705 a) increases by fg, the flow ratein the expanding channel increases by fq. Similarly, when theconductance of a contracting channel (e.g., channel 705 b) decreases byfg, the flow rate in the contracting channel decreases by fq.

The fluidic conductance of each channel in the array is a function ofits dimensions and the fluid viscosity. In the array shown in FIG. 7,each channel is assumed to have a rectangular cross-section andtherefore has conductance described by

$g \approx {( \frac{h^{4}}{12\; \eta \; l\; \alpha} )( {1 - {0.63\; \alpha}} )}$

Here, η is fluid viscosity, l is channel length, w is channel width, his channel height, and a=h/w. A more accurate infinite series-basedformula is also available (Tanyeri et al., “A microfluidic-basedhydrodynamic trap: Design and implementation (Supplementary Material).”Lab on a Chip (2011).) Computational modeling or empirical methods canbe used to determine the conductance of more complex channel geometries.(Note that in this description it is simpler to focus on fluidicconductance, g, rather than fluidic resistance, R. The two quantitiesare simply related by g=1/R.)

After the fluidic conductance of the interior channel has beendetermined, the fluidic conductance of the i^(th) unit of expandingchannel, g_(e,i), may be expressed as

g_(e,i)=ifg

That is, the fluidic conductance of the first unit is fg, and thefluidic conductance of each successive unit increases by fg. Noting thatthe flow rate in the i^(th) unit of expanding channel, q_(e,i), isrelated to the conductance by q_(e,i)=pg_(e,i) and that q=pg, the flowrate in the i^(th) unit of expanding channel may be expressed as

q_(e,i)=ifq

Thus, the flow rate in the first unit is fq, and the flow rate in eachsuccessive unit increases by fq.

The fluidic conductance of the i^(th) unit of contracting channel,g_(c,i), may be expressed as

g _(c,i)=2g−ifg

That is, the fluidic conductance of the first unit is 2g−fg, and thefluidic conductance of each successive unit decreases by fg. Noting thatthe flow rate of the i^(th) unit of contracting channel, q_(c,i), isrelated to the conductance by q_(c,i)=pg_(c,i) and that q=pg, the flowrate in the i^(th) unit of expanding channel may be expressed as

q _(c,i)=2q−ifq

Thus, the flow rate in the first unit is 2q−fq, and the flow rate ineach successive unit decreases by fq.

The width of the expanding channel, w_(e,i), is chosen to give therequired g_(e,i), and the width of the contracting channel, w_(c,i), ischosen to give the required g_(c,i). In practice, these widths may bedetermined by evaluating fluidic conductance (using the above formula)across a wide range of channel widths and then interpolating to find thechannel width that gives the desired channel conductance.

The number of units needed to increase the flow in the expanding channelto q and decrease the flow in the contracting channel to q is n=1/f.Thus, in the n^(th) unit, the flow rates in all channels are equal:q_(e,n)=q_(c,n)=q.

After n units, the array may be “reset.” For instance, FIG. 8 is aschematic that illustrates a top view of an example particle sortingregion 800, similar to the region 700 shown in FIG. 7, in which a“reset” region is introduced after n preceding units of islandstructures 710. At the reset, the contracting channel 705 b and theadjacent interior channel 703 combine to form a new contracting channel709 b, the expanding channel 705 a becomes an interior channel, and anew expanding channel 709 a is introduced.

The unit length, width, shift, flow speed, and particle size are thefactors that most significantly impact performance of the device.Briefly, the impact of each is as follows:

-   -   The unit length determines the distance (and time) over which        the inertial lift force acts on a particle, and therefore        determines the lateral distance that a particle migrates per        unit. For a particle to be retained, the unit must be long        enough for the particle to escape from the fluid that will be        shifted at the next opening between islands.    -   The flow speed also impacts the magnitude of the inertial lift        force and the lateral distance that a particle migrates per        unit. The migration (lateral) distance per longitudinal distance        is approximately proportional to the flow speed. For a particle        to be retained, the flow speed must be fast enough for the        particle to escape from the fluid that will be shifted at the        next opening between islands.    -   The shift does not affect particle migration directly, but        rather determines how far (i.e., across what fraction of the        fluid) a particle must migrate to escape the fluid that will be        shifted at the next opening between islands. The larger the        shift, the farther a particle must migrate.    -   The unit width affects performance in two ways. First, the width        (and height) of the unit affect the magnitude of the inertial        lift force acting on a particle, with the force decreasing as        unit width increases. Second, the width relates the shift to the        distance a particle must migrate. In other words, for a given        shift, the larger the unit width, the farther a particle must        migrate to escape the fluid that will be shifted at the next        opening between islands.    -   The magnitude of the inertial lift force is strongly dependent        on particle size, with the force increasing dramatically with        particle size (D. Di Carlo. “Inertial Microfluidics.” Lab on a        Chip (2009).). As a result, larger particles laterally migrate        farther per unit than smaller particles. It is this difference        in migration rates that enables size-based sorting of particles.

For exchanging fluid between channels, the above factors may be chosento ensure that the particles of interest are retained within the rows ofthe array. For size-based particle sorting applications, the abovefactors may be chosen such that a subpopulation of larger particles isretained within the rows while a subpopulation of smaller particles isnot.

For instance, the following set of parameters can be used to debulkblood (i.e., separate white blood cells (WBCs) from red blood cells(RBCs) and platelets): unit length of about 200 μm, a unit width ofabout 50 μm, a unit depth of about 52 μm, about 3.0% shift, and a flowrate of about 80 μL/min per row (0.51 m/s average flow speed). This setof parameters can be highly effective in isolating WBCs with minimalcarryover of RBCs and platelets. In this case, white blood cells arespherical and typically >8 μm diameter. Red blood cells are disk shapedwith a ˜7 μm diameter and ˜1.5 μm thickness (and are therefore expectedto behave like spheres of intermediate size). Platelets are disk shapedwith 3 μm diameter.

The islands have 200 μm length (i.e., same as unit length) and 50 μmwidth. The purpose of the islands is simply to separate the channels soas to establish the appropriate flow conditions within the device. Assuch, the width of the islands is not of particular functionalimportance. The islands could be made somewhat narrower or wider withoutsignificantly affecting the performance of the device.

However, the width of the islands does impact ease of manufacturing.Ease of manufacturing is largely determined by the aspect ratio (heightdivided by width) of structures within a microfluidic device, withsmaller aspect ratio devices being easier to manufacture at low cost andwith high manufacturing yield. We can define the aspect ratio in twoways. The minimum aspect ratio is the structure height, h, divided bythe minimum structure width, w_(min). The overall aspect ratio is thestructure height, h, divided by the diameter, D, of a circle with thesame area as the structure. Here, D can be expressed as D=√(4A/π), whereA is the area of the structure.

Because the islands in the example above have 50 μm width and 52 μmheight, they have a minimum aspect ratio of 1.04 and an overall aspectratio of 0.46. This may enable straightforward fabrication of moldedPDMS and epoxy devices, as well as injection molded plastic devices.Thus, the device is not only extremely useful from a functionalperspective, but it is also fundamentally scalable and economical from acommercial perspective. Furthermore, the set of device parameters listedabove can be modified to sort particles of other sizes.

A microfluidic device that is configured to shift particles of a givensize can, in some implementations, be scaled to effectively shiftparticles of a different size. For instance, for a device that employsinertial lift forces to shift particles across fluid streamlines, onecan scale the dimensions of the particle shifting area with particlesize and alter the flow conditions, so long as the value of the particleReynolds number, R_(p), is preserved. The particle Reynolds number canbe expressed as:

$R_{p} = \frac{U_{m}a^{2}}{v\; D_{h}}$

where U_(m) is the maximum channel velocity, a is the particle diameter,v is the kinematic viscosity of the fluid, and D_(h) is the hydraulicdiameter of the channel. For channels of rectangular cross-section withwidth w and height h, D_(h) can be expressed as (2wh)/(w+h), where h isthe channel height and w is the channel width. For example, consider aShifting Area 1 that effectively shifts particles of size a. One methodof designing a Shifting Area 2 that effectively shifts particles of size2a is to scale all dimensions of Shifting Area 1 by a factor of 2 (i.e.,double the length, width, and height of all features). To maintain thesame R_(p) in Shifting Area 2, the maximum channel velocity U_(m) mustbe decreased by a factor of 2.

Other methods of scaling the dimensions of particle shifting areas andflow conditions with particle size are also possible.

For sorting devices with straight channels that rely on inertial liftforces to shift particles across streamlines, the following providesdevice design and operation guidelines:

First, as described in “Inertial Microfluidics,” Di Carlo, Lab Chip (9),3038-3046, 2009 (incorporated herein by reference in its entirety), theratio of the lateral (across channel) particle velocity U_(y) to thelongitudinal (in direction of fluid flow) velocity U_(z) is proportionalto the particle Reynolds number R_(p) and can be expressed as:

${\frac{U_{y}}{U_{z}} \propto R_{p}} = \frac{U_{m}a^{2}}{v\; D_{h}}$

Here U_(m) is the maximum channel velocity, a is the particle diameter,v is the kinematic viscosity of the fluid, and D_(h) is the hydraulicdiameter of the channel. (For channels of rectangular cross-section withwidth w and height h, D_(h)=(2wh)/(w+h).) A goal of the particle sortingdevice described herein, in some implementations, is to use inertiallift forces to efficiently move particles across streamlines (i.e.,maximize U_(y)/U_(z)). For that purpose, it is recommended that thechannel dimensions and flow conditions be selected so as to maximizeparticle Reynolds number R_(p) in the particle channel to the extentpermitted by other practical constraints, such as operating pressure.Throughout the device, the particle Reynolds number R_(p) in theparticle channel should ideally be greater than about 0.01, though itmay be much larger than this, possibly greater than 100. R_(p)approximately equal to 1 is a good intermediate target.

For a given particle diameter a and kinematic viscosity v, a targetparticle Reynolds number R_(p) can be achieved through many differentcombinations of channel dimensions and channel velocities. One possiblestrategy for increasing R_(p) would be to select a very small (relativeto a) hydraulic diameter D_(h). However, channel resistance has aquartic dependence on D_(h), and choosing an unnecessarily small D_(h)comes at the cost of highly increased operating pressure. On thecontrary, the operating pressure scales linearly with channel velocityU_(m), so a good alternative strategy is to design a device with amodest hydraulic diameter D_(h) and then increase channel velocity U_(m)(and therefore R_(p)) at the time of operation as needed to achieve highyield of particles. For a channel with square cross-section, such thatD_(h)=w=h, a value of D_(h) approximately five times the particlediameter a is a reasonable choice: D_(h)=5a.

Second, the length of the openings (in the longitudinal direction)between islands is preferably, though not necessarily, greater thanabout a and less than or equal to about w. If the length of the openingis less than a, the opening may clog with particles, thereby disruptingflow through the opening. An opening with length approximately equal tow is unlikely to clog with particles and provides adequate room forfluid to cross between islands to the adjacent channel. An opening witha length greater than w will work but provides no particular benefit andcomes at the cost of wasted space.

Third, the length of the islands l is preferably greater than or equalto the length of the openings between islands. Because particlesexperience inertial lift forces as they travel alongside islands, not atthe openings, particles should travel most of their longitudinaldistance alongside islands, rather than across openings between islands.Put another way, if the length of islands and the length of the openingsbetween islands are equal, then particles experience inertial liftforces along just 50% of the distance they travel. On the other hand, ifthe length of the islands is four times the length of the openings, thenparticles experience inertial lift forces along 80% of the distance theytravel.

A loose upper limit on the length of islands l is the length requiredfor particles to migrate to equilibrium focusing positions. Anyadditional channel length beyond what is required for particles to reachequilibrium does not contribute to shifting particles acrossstreamlines. A formula for the channel length L_(f) required forparticles to reach equilibrium is given in “Inertial Microfluidics,” DiCarlo, Lab Chip (9), 3038-3046, 2009 and can be expressed as:

$L_{f} = \frac{\pi \; \mu \; w^{2}}{\rho \; U_{m}a^{2}f_{L}}$

Here μ is dynamic viscosity, w is channel width, ρ is fluid density,U_(m) is the maximum channel velocity, a is the particle diameter, andf_(L) is a dimensionless constant ranging from about 0.02 to 0.05 forchannels with aspect ratios (h/w) ranging from about 2 to 0.5. WhileL_(f) provides an upper bound, it is a loose upper bound and exceeds theoptimal length of islands l. This is because the lift force on particlesis very strong near the channel wall (proportional to a⁶), but falls offsharply with distance from the wall (proportional to a³ near the centerof the channel). Thus, a sorting device will more efficiently shiftparticles across streamlines if the particles are kept near the channelwall by using an island length l that is significantly less than L_(f).

Given these considerations, a reasonable intermediate value for theisland length is about l=4w. This is an approximate value andnecessarily depends on the values selected for other parameters, such asthe fluid shift f_(s).

Fourth, the fluid shift f_(s) should be greater than 0.2% and ideallygreater than 1.0%. If the fluid shift is small, e.g., 0.1%, then thetotal number of shifts (units) needed to shift particles across thewidth of the sorting array is very large and the device itself musttherefore be very long. Provided the maximum channel velocity U_(m) issufficiently high to place the particle Reynolds number R_(p) in theprescribed range, an extremely small shift, e.g., 0.1%, should not benecessary. Depending on the maximum channel velocity U_(m), a fluidshift f_(s) in the range of about 1% to 5% should perform well for adevice designed and operated as outlined here.

For any given device design and particle size a, the final parameterchoice is the device operating flow rate, which directly determines themaximum channel velocity U_(m) and the particle Reynolds number R_(p) inthe particle channel. For a device designed as outlined, there will be alower end flow rate that provides good performance. Below this thresholdflow rate, the inertial lift forces will be insufficient to shiftparticles far enough from the island wall to avoid being shifted withthe fluid through the islands, thus resulting in low yield of particles.While the formulas provided here enable one to make rough estimates ofthe threshold flow rate, the most accurate and relevant method ofdetermining the threshold flow rate is empirically.

If the sorting device is to be used to fractionate particles based onsize (i.e., there are two or more populations of particles withdifferent size), then the operating flow rate should be selected suchthat the inertial lift forces are sufficient to shift the largeparticles without shifting the small particles.

The design and operating parameter guidelines described here have beenfound to work well for cell sorting devices. However, other design andoptimization strategies may also result in effective, high performanceparticle sorting devices.

Microfluidic Device Dimensions

For generally spherical particles being transported through amicrofluidic device having at least two channels separated by an arrayof island structures, with gaps between adjacent islands (see, e.g.,FIG. 1), the depth (e.g., as measured along the x-direction in FIG. 1)and width (e.g., as measured along the y-direction in FIG. 1) of eachmicrofluidic channel is preferably in the range of about 2 times toabout 50 times the diameter of a single particle. With respect to therigid structures that form the gaps through which fluid is extracted,the width of the structures may be up to about 10 times the width of thea single microfluidic channel, whereas the length of the structures maybe between about 0.25 times the channel width up to about 50 times thechannel width.

As an example, for a generally spherical particle having a diameter ofabout 8 microns, a microfluidic device having two microfluidic channelsseparated by an array of rigid structures similar to the configurationshown in FIG. 1 may have the following parameters: each microfluidicchannel and island structure may have a depth of about 50 μm, eachmicrofluidic channel may have a width of about 50 μm, each islandstructure may have a width of about 50 μm, each island structure mayhave a length of about 200 μm.

Other examples of dimensions are set forth as follows.

For instance, the distance between the outer walls of the areacontaining the different fluid flow regions, i.e., as measuredtransverse to the fluid flow direction, can be configured to be betweenabout 1 μm to about 100 mm (e.g., about 10 μm, about 50 μm, about 100μm, about 500 μm, about 1 mm, about 50 mm, about 10 mm, or about 50 mm).Other sizes are possible as well. The width of each fluid flow region,measured transverse to the fluid flow direction, can be configured to bebetween about 1 μm to about 10 mm (e.g., about 50 μm, about 100 μm,about 250 μm, about 500 μm, about 750 μm, about 1 mm, or about 5 mm).Other distances are possible as well.

The length of the gaps/openings between the island structures, asmeasured along the fluid flow direction (e.g., along the z-direction inFIG. 1), can be configured to be between about 500 nm to about 1000 μm(e.g., about 1 μm, about 2 μm, about 54 μm, about 106 μm, about 50 μm,about 100 μm, about 200 μm, about 500 μm, or about 750 μm). In someimplementations, the length of each successive opening is greater thanor less than the length of the last opening. For example, in a channelconfigured to have a decreasing fluidic resistance along the fluidpathway, each successive opening may be larger so that a greater amountof fluid is extracted through the opening. The island structures thatseparate different fluid flow regions can be configured to have a lengthbetween about 10 nm to about 1000 μm, and a width between about 10 nm toabout 1000 μm. Other dimensions for the gaps and island structures arepossible as well.

The height of the fluid flow regions and the island structures withinthe particle shifting area (e.g., as measured along the x-direction inFIG. 1) are within the range of approximately 100 nm to approximately 10mm. For example, the height of the channel can be about 500 nm, about 1μm, about 5 μm, about 10 μm, about 50 μm, about 100 μm, about 500 μm,about 750 μm, about 1 mm, or about 5 mm. Other heights are possible aswell. The microfluidic flow regions can have a cross-sectional area thatfalls, e.g., within the range of about 1 μm² to about 100 mm².

Microfluidic Systems

In some implementations, the particle shifting areas of the microfluidicdevices described herein are part of a larger, optional, microfluidicsystem having a network of microfluidic channels. Such microfluidicsystems can be used to facilitate control, manipulation (e.g., sorting,separation, segregation, mixing, focusing, concentration), and isolationof liquids and/or particles from a complex parent specimen. During theisolation process, microfluidic elements provide vital functions, forexample, handling of biological fluids or reproducible mixing ofparticles with samples.

For example, the microfluidic system may include additional areas forsorting particles according to size and/or shape using other techniquesdifferent from inertial lift forces. These other techniques can include,for example, deterministic lateral displacement. The additional areasmay employ an array of a network of gaps, in which a fluid passingthrough a gap is divided unequally into subsequent gaps. The arrayincludes a network of gaps arranged such that fluid passing through agap is divided unequally, even though the gaps may be identical indimensions. In contrast to the techniques described herein forseparating particles based on a combination of inertial lift forces andfluid extraction, deterministic lateral displacement relies on bumpingthat occurs when the particle comes into direct contact with postsforming the gaps. The flow of the fluid is aligned at a small angle(flow angle) with respect to a line-of-sight of the array. Particleswithin the fluid having a fluidic size larger than a critical sizemigrate along the line-of-sight in the array, whereas those having afluidic size smaller than the critical size follow the flow in adifferent direction. Flow in the device generally occurs under laminarflow conditions. In the device, particles of different shapes may behaveas if they have different sizes. For example, lymphocytes are spheres of·5 μm diameter, and erythrocytes are biconcave disks of ˜7 μm diameter,and ˜1.5 μm thick. The long axis of erythrocytes (diameter) is largerthan that of the lymphocytes, but the short axis (thickness) is smaller.If erythrocytes align their long axes to a flow when driven through anarray of posts by the flow, their fluidic size is effectively theirthickness (˜1.5 μm), which is smaller than lymphocytes. When anerythrocyte is driven through an array of posts by a fluidic flow, ittends to align its long axis to the flow and behave like a ˜1.5 μm-wideparticle, which is effectively “smaller” than lymphocytes. The area fordeterministic lateral displacement may therefore separate cellsaccording to their shapes, although the volumes of the cells could bethe same. In addition, particles having different deformability behaveas if they have different sizes. For example, two particles having theundeformed shape may be separated by deterministic lateral displacement,as the particle with the greater deformability may deform when it comesinto contact with an obstacle in the array and change shape. Thus,separation in the device may be achieved based on any parameter thataffects hydrodynamic size including the physical dimensions, the shape,and the deformability of the particle.

Additional information about microfluidic channel networks and theirfabrication can be found, for example, in U.S. Patent App. PublicationNo. 2011/0091987, U.S. Pat. Nos. 8,021,614, and 8,186,913, each of whichis disclosed herein by reference in its entirety.

In some implementations, a microfluidic system includes components forpreparing a particle carrying fluid sample prior to introducing thefluid sample into a particle shifting area. For instance, FIG. 6A is aschematic that illustrates a top view of an example of a microfluidicsystem 600 that includes a particle shifting area 601 (labeled “SortingUnits”), similar to the particle shifting area shown in FIG. 1. Otherconfigurations may be used as the particle shifting area, such as any ofthe configurations shown in FIGS. 2-5. For configurations that includetwo or more inlet channels, the system 600 may include an additionalfluid source or sources for those inlets.

FIG. 6B is a schematic that illustrates an enlarged view of the particlesorting region 601. As shown in the enlarged view, the region 601includes a first microfluidic channel 650 and a second microfluidicchannel 652 that extends along the first microfluidic channel 650. Thesecond and first microfluidic channels are separated from one another byan array of island structures 654, in which each island in the array isseparated by an adjacent island in the array by a gap or opening 656that fluidly couples the first microfluidic channel 650 to the secondmicrofluidic channel 652. The fluidic resistance of the area 601 changesalong a longitudinal direction of the area 601(e.g., in the direction offluid propagation) such that fluid flowing in the first microfluidicchannel 650 passes through the openings 656. Inertial lift forces causeparticles flowing within the first microfluidic channel 650 near theisland structures to cross streamlines so they do not follow the fluidthat passes into the second microfluidic channel 652.

The system 600 additionally includes a filter section 603 (labeled“Filter”) and a particle focusing section 605 (labeled “FocusingChannel”) upstream from the particle shifting area 601. The filtersection 603 includes an arrangement of multiple different-sized poststructures. Based on the arrangement of the structures, the filtersection 603 is configured to filter particles contained in an incomingfluid according to the particle size (e.g., average diameter), such thatonly particles of a pre-defined size or less are able to pass to thenext stage of the system 600. For instance, for complex matrices, suchas bone marrow aspirate, the filter section 603 may be configured toremove bone chips and fibrin clots to improve the efficiency ofenhancing concentration downstream. In an example arrangement, thefilter section 603 may include an array of posts having a pillar sizeand array offset designed to deflect particles above a certain size,thereby separating them from the main suspension. Typically, the sizelimit is determined based on the maximum particle size that can passthrough later stages of the system 600. For example, the filter 603 maybe configured to filter/block passage of particles that have an averagediameter greater than 50%, greater than 60%, greater than 70%, greaterthan 80% or greater than 90% of the minimum width of a channel in theparticle shifting area 601.

The filter section 603 is fluidly coupled to the particle focusingsection 605. The particle focusing section 605 is configured topre-focus particles exiting the filter section 603 to a desired fluidstreamline position, before the particles are provided to the particleshifting area 601. An advantage of pre-focusing the particles is that itreduces the distribution of particles across the channel width to anarrow lateral extent. The focused line of particles then can berepositioned so that the probability of the particles inadvertentlyentering the wrong channel within the particle sorting area 601 isreduced (e.g., to avoid the particles entering the second microfluidicchannel 652 instead of the first microfluidic channel 650). Pre-focusingcan be achieved using inertial focusing techniques. Further details ofinertial focusing can be found, for example, in U.S. Pat. No. 8,186,913,which is incorporated herein by reference in its entirety.

Once the particles have been sorted in the particle shifting area 601,the sorted particles may be coupled to separate processing regions ofthe microfluidic system 600 or removed from the system 600 foradditional processing and/or analysis. For example, the second channelof the particle shifting area 601 is coupled to a first outlet 607,whereas the second channel of the particle shifting area 601 is coupledto a second outlet 609.

External Forces

Other functionality may be added to the microfluidic system to enhancethe focusing, concentrating, separating, and/or mixing of particles. Forinstance, in some implementations, additional forces may be introducedwhich result in target specific modification of particle flow. Theadditional force may include, for example, magnetic forces, acousticforces, gravitational/centrifugal forces, electrical forces, and/orinertial forces.

Fabrication of Microfluidic Devices

A process for fabricating a microfluidic device according to the presentdisclosure is set forth as follows. A substrate layer is first provided.The substrate layer can include, e.g., glass, plastic or silicon wafer.An optional thin film layer (e.g., SiO₂) can be formed on a surface ofthe substrate layer using, for example, thermal or electron beamdeposition. The substrate and optional thin film layer provide a base onwhich microfluidic regions may be formed. The thickness of the substratecan fall within the range of approximately 500 μm to approximately 10mm. For example, the thickness of the substrate 210 can be 600 μm, 750μm, 900 μm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, or 9 mm.Other thicknesses are possible as well.

After providing the substrate layer, the microfluidic channels formedabove the substrate layer. The microfluidic channels include thedifferent fluid flow pathways of the particle shifting area, as well asthe other microfluidic components of the system, including any filteringsections, inertial focusing sections, and magnetophoresis sections.Microfluidic channels for other processing and analysis components of amicrofluidic device also may be used. The microfluidic channels andcover are formed by depositing a polymer (e.g., polydimethylsiloxane(PDMS), polymethylmethacrylate (PMMA), polycarbonate (PC), or cycloolefin polymer (COP)) in a mold that defines the fluidic channelregions. The polymer, once cured, then is transferred and bonded to asurface of the substrate layer. For example, PDMS can be first pouredinto a mold (e.g., an SU-8 mold fabricated with two stepphotolithography (MicroChem)) that defines the microfluidic network ofchannels. The PDMS then is cured (e.g., heating at 65° C. for about 3hours). Prior to transferring the solid PDMS structure to the device,the surface of the substrate layer is treated with O₂ plasma to enhancebonding. Alternatively, the microfluidic channels and cover can befabricated in other materials such as glass or silicon.

Applications

The new microfluidic techniques and devices described herein can be usedin various different applications.

Centrifugation Replacement

The particle shifting techniques and devices disclosed herein can beused as replacements for centrifugation. In general, centrifugation isunderstood to include the concentrating of sub-components within a fluidthrough the application of centrifugal forces to the fluid. Typically,this process requires devices that have moving parts, which are prone towear and breakage. Moreover, the moving parts require complex and costlyfabrication processes. Another problem with centrifugation is that it isa process typically applied in a closed system, i.e., centrifugationrequires manually transferring samples to and from a centrifuge.

In contrast, the presently disclosed techniques are capable ofsubstantially increasing the concentration of fluid components usingrelatively simple micro-structures without the need for moving parts.The techniques can be implemented as part of a single open microfluidicsystem, such that fluid samples may be transferred to or from theparticle shifting area without manual interference. Additionally,particle shifting can be extended to devices requiring large throughput(i.e., volume rate of fluid that can be processed) without a substantialdegradation in particle separation efficiency. For example, the devicesdisclosed herein may be configured to enable up to 10, 25, 50, 75, 100,250, 500, 1000, 5000, or 10000 μl/min of fluid flow. Other flow ratesare also possible. For instance, using device 100 in FIG. 1 as anexample, if the second and first microfluidic channels 106, 108 havedepths of approximately 50 μm and widths of approximately 50 μm, thedevice 100 may be capable of achieving a combined sample flow rate of upto about 5 mL/min. Varying the channel sizes may alter the maximumvolumetric flow rate of which the device is capable. Additionally, or asan alternative, the volumetric flow rate may be adjusted by varying thelength of the island structures (see section “Microfluidic Device DesignParameters” above). Furthermore, multiplexing multiple channels (e.g.,operating multiple particle sorting regions in parallel) may enable evenhigher rates of flow.

In some implementations, the particle shifting techniques allowseparation of particles based on size. For instance, in a fluid samplecontaining two different sized particles, the particle sorting regiondescribed according to the present disclosure may be used to separatethe larger particles from the smaller particles (e.g., by siphoning thesmaller particles from the fluid sample into an adjacent microfluidicchannel while using inertial lift forces to maintain the largerparticles in the original microfluidic channel). In another example, afluid sample may include particles of three or more different sizes, inwhich the particle sorting region is designed to sort the particles intodifferent regions based on their different sizes.

Thus, in certain implementations, the particle shifting techniques mayprovide substantial cost and time saving advantages over traditionalcentrifugation processes. Examples of applications where a microfluidicreplacement for a centrifuge device may be useful include bone marrowand urine analysis.

Detecting Infectious Agents

In addition, the particle shifting techniques disclosed herein can beused as part of a research platform to study analytes of interest (e.g.,proteins, cells, bacteria, pathogens, and DNA) or as part of adiagnostic assay for diagnosing potential disease states or infectiousagents in a patient. By separating and focusing particles within a fluidsample, the microfluidic device described herein may be used to measuremany different biological targets, including small molecules, proteins,nucleic acids, pathogens, and cancer cells. Further examples aredescribed below.

Rare Cell Detection

The microfluidic device and methods described herein may be used todetect rare cells, such as circulating tumor cells (CTC) in a bloodsample or fetal cells in blood samples of pregnant females. For example,the concentration of primary tumor cells or CTCs can be enhanced in ablood sample for rapid and comprehensive profiling of cancers. Bycombining the particle deflection techniques described herein withmagnetophoresis, different types of cells can be detected (e.g.,circulating endothelial cells for heart disease). Thus, the microfluidicdevice may be used as a powerful diagnostic and prognostic tool. Thetargeted and detected cells could be cancer cells, stem cells, immunecells, white blood cells or other cells including, for example,circulating endothelial cells (using an antibody to an epithelial cellsurface marker, e.g., the Epithelial Cell Adhesion Molecule (EpCAM)), orcirculating tumor cells (using an antibody to a cancer cell surfacemarker, e.g., the Melanoma Cell Adhesion molecule (CD146)). The systemsand methods also can be used to detect CTC clusters, small molecules,proteins, nucleic acids, or pathogens.

Fluid Exchange

The microfluidic device and methods described herein may be used toshift cells from one carrier fluid to another carrier fluid. Forexample, the particle shifting techniques disclosed could be used toshift cells into or out of a fluid stream containing reagents, such asdrugs, antibodies, cellular stains, magnetic beads, cryoprotectants,lysing reagents, and/or other analytes.

A single particle shifting region could contain many parallel fluidstreams (from many inlets) through which a shifted cell would pass. Forexample, white blood cells could be shifted from a blood stream into astream containing staining reagents and then into a buffer stream.

In bioprocessing and related fields, the devices and techniquesdescribed may be used to enable sterile, continuous transfer of cellsfrom old media (containing waste products) into fresh growth media.Similarly, extracellular fluids and cellular products (e.g., antibodies,proteins, sugars, lipids, biopharmaceuticals, alcohols, and variouschemicals) may be extracted from a bioreactor in a sterile, continuousmanner while cells are retained within the bioreactor.

Separating and Analyzing Cells

The microfluidic device and methods described herein may be used tofractionate cells based on biophysical properties, such as size. Forexample, the device and methods may be used to fractionate blood intoseparate platelet, red blood cell, and leukocyte streams. In anotherexample, the device and methods may be used to fractionate leukocytesinto its separate lymphocyte, monocyte, and granulocyte streams.

The streams of fractionated cells may be isolated by routing them intoseparate fluid outlets. Alternatively, the streams of cells may bedetected and analyzed in real-time (e.g., using optical techniques) todetermine the number of cells in each stream or properties, such as sizeor granularity, of the cells in each stream.

Techniques may be used to alter cells or their carrier fluid before orduring sorting to facilitate their fractionation and/or analysis. Forexample, large beads may be bound to a specific cell type increase theeffective size of that cell type. Controlled cell aggregation may alsobe used to increase the effective size of cells. The temperature,density, viscosity, elasticity, pH, osmotic, and other properties of thefluid may be changed to either directly affect the sorting process(e.g., inertial effects are viscosity dependent) or indirectly affectthe sorting process by altering the properties of cells (e.g., osmoticswelling or shrinking).

Fluid Sterilization and Cleansing

The microfluidic device and methods described herein may be used toremove pathogens, pollutants, and other particular contaminants fromfluids. By shifting contaminants across fluid streamlines, contaminantsmay be removed from a fluid sample and collected as a separate wastestream.

Harvesting Algae for Biofuels

Harvesting algae from growth media is a major expense in the productionof biofuels because algae grow in very dilute suspensions at nearneutral buoyancy, making efficient extraction and concentration of algalbiomass difficult. The microfluidic device and methods described hereincan provide an efficient means of harvesting algae that does not dependon either density or filtration. The devices and techniques describedenable the algae in a growth tank to be extracted from the growth mediaand concentrated to a high volume density. This could be done either asa single step or as part of a continuous process. Additionally, becausethe devices described herein can sort cells in a size-dependent manner,they may be designed to sort and concentrate only the larger algae thathave reached maturity, returning smaller, immature algae to the tank.

Micro Heat Exchangers

The devices and methods described herein can be used to process not onlyliquid flows, but also gaseous and multi-phase flows. One exampleapplication of interest is in high efficiency heat exchangers forintegrated circuits. The high power density in microchips necessitatesefficient removal of waste heat. Such cooling becomes increasinglydifficult as microchips are stacked, decreasing the overall surface tovolume ratio. Liquid cooling, in which heat passes from a heat sourceinto a flowing liquid, is one approach for increasing the cooling rateof microchips. Such cooling can be particularly efficient near theboiling point of the liquid, as considerable energy is absorbed in theliquid to vapor phase change. However, accumulation of vapor (bubbles)at the heat exchange surface dramatically reduces heat flux. The devicesand techniques described herein could be used to sweep bubbles away fromthe heat exchange surface, maximizing heat absorbance by the liquid (viaphase change) while minimizing the fraction of the surface in contactwith vapor. For this application, one side of the sorting module wouldcontact the microchip heat source. As liquid flowing through moduleabsorbed heat, bubbles would form on the heat exchange surface and thenbe swept into the flow (by fluid drag) upon reaching a critical size.The sorting array would then direct these bubbles across the sortingmodule and away from the heat exchange surface, thereby maximizing heatflux from the microchip to the cooling liquid.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1 Debulking

Debulking is the removal of plasma, red blood cells (RBCs), platelets,and other small components (e.g., magnetic beads) from nucleated cells(e.g., white blood cells (WBCs)) in blood and other complex fluids, suchas bone marrow aspirate (BMA). This is typically achieved by densitycentrifugation, which separates blood into layers by density. However,in the following examples, we describe the use of a sorting device thatrelies on inertial lift forces to debulk blood.

Device Fabrication

For fabricating the microfluidic device, standard SU8 photolithographyand soft lithography techniques were used to fabricate the master moldand the PDMS microchannels, respectively. Briefly, negative photoresistSU8-50 (Microchem Corp, Massachusetts) was spun at 2850 RPM to athickness of approximately 50 μm, exposed to ultraviolet light through amylar emulsion printed photomask (Fineline Imaging, Colorado) thatdefines the microfluidic network of channels, and developed in BTS-220SU8-Developer (J.T. Baker, New Jersey) to form a raised mold. A 10:1ratio mixture of Sylgard 184 Elastomer base and curing agent (DowCorning, Michigan) was then poured over the raised mold, allowed to curein an oven at 65° C. for 8 hours and then removed from the SU8 mastermold to form the microfluidic device cover having the patternedchannels. Inlet and outlet holes to the channels were punched usingcustom sharpened needle tips. The devices were then cleaned ofparticulate using low-residue tape and oxygen plasma bonded topre-cleaned 1 mm thick glass microscope slides.

The devices had the following parameters: 200 μm unit length, 50 μm unitwidth, 52 μm unit depth (according to the unit length design discussedabove with respect to FIG. 7). The island structures had a length of 200μm (i.e., same as unit length) and a width of 50 μm.

Sample Preparation

The performance of the debulking device was evaluated across a largenumber (n=63) independent experiments. In each experiment, ≥2 mL offresh whole blood with either EDTA or ACD anticoagulant was diluted 1:1with PBS (1×) with 1% F68 Pluronic.

Experimental Procedure and Results

For each run, the blood sample was driven into the device using asyringe pump operating at 60 μL/min. The buffer coflow (PBS (1×) with 1%F68 Pluronic) was driven into the device using a syringe pump operatingat 272 μL/min. The compositions of the input, product, and waste wereanalyzed using a hematology analyzer (Sysmex KX21N). Manual countingusing Neubaur and Nageotte chambers was also performed on the productand waste to ensure accurate data for cell types present in lowconcentrations (specifically WBCs in the waste and RBCs and platelets inthe product).

The data resulting from the experiments is summarized in FIGS. 9A-9D.The median WBC yield is 85.9% and the median neutrophil yield is 93.4%.That the neutrophil yield is somewhat higher than the overall WBC yieldis consistent with the fact that neutrophils are the largest WBCsubpopulation in physical size. The purity of the product is excellent.The median carryover of RBCs (i.e., percentage of input RBCs that end upin the product) is just 0.0054%, and the median carryover of plateletsis just 0.027%, indicating very few RBCs and platelets remain in thesame fluid streams as the WBCs and neutrophils. In contrast to someother approaches to microfluidic fraction, the approach presented hereis sensitive to the flow rate in the device. This is because theinertial lift force is strongly dependent on flow speed. In the array,the relevant flow rate is the flow rate per row.

Additional experiments were performed where the flow rate was varied toevaluate the effect on debulking. FIGS. 10A-10B show the WBC yieldplotted against the flow rate per row. At low flow rates (<10 μL/min),the inertial lift force is too weak to move WBCs out of the shiftedfluid streams, and therefore the yield is ˜0%. As the flow rateincreases toward 60μL/min, the inertial lift force increases and alarger percentage of WBCs escape the shifted fluid streams and therebyreach the product, increasing the yield. At the highest flow rates(>60μL/min), the inertial lift force is large enough to vector the largemajority of WBCs across the array and into the product, with the WBCyield plateauing at ˜85%. For reference, the flow rate per row in thedebulking experiment of FIG. 9 was 80 μL/min.

The strong dependence of WBC yield on flow rate suggests that it maypossible to control the fractionation size threshold by modulating theflow rate per row. For any given flow rate, the inertial lift force on aparticle depends on its size. Therefore, we would expect that the curveshown in FIGS. 10A-10B would shift left for larger cells (orsubpopulations of larger cells within the WBC population) and that thecurve would shift right for smaller cells (or subpopulations of smallercells within the WBC population). Operating at a flow rate per row largeenough to move large cells (e.g., neutrophils) across the array but notlarge enough to move small cells (e.g., lymphocytes) across the arraymay be a way of isolating particular subpopulations of cells with highpurity.

Example 2 Evaluating Impact of Particle Size, Fluid Flow Rate, and FluidShift

The impact of the design and process factors on device performance canbe illustrated using two experiments. The first uses fluorescent beadsto show the impact of particles size and flow rate per row on yield, andthe second uses white blood cells (WBCs) to show the impact of the shifton yield. The device used for the experiments was fabricated accordingto the same procedure as set forth above in Example 1.

For the first experiment, fluorescent beads of several different sizeswere used across a range of flow rates per row. Each bead size wastested independently. In each case, a sample including beads suspendedin buffer (PBS (1×) with 1% F68 Pluronic), entered the device alongsidea stream of buffer.

FIG. 11 shows the yield of the different sized fluorescent beads acrossthe different flow rates. The total flow rate (sample+buffer) was chosento give the indicated flow rate per row, and the relative input flowrates of the sample and buffer were chosen such that 18% of the totalinput flow was sample. At the end of the device, particles that hadmigrated downward exited the device through the product channel and werecollected in a vial. Particles that remained at the top of the arrayexited the device through the waste channel and were collected in aseparate vial. The volumes of the product and waste vials were measuredby mass, and the concentrations of the particles were determined usingstandard Neubauer and Nageotte counting chambers. The relative yield wascalculated as the fraction of output beads in the product.

A few trends stand out in the resulting data. First, for any given beadsize, the yield increases with flow rate. This is due to the increase inthe inertial lift force with flow speed. Second, for any given flowrate, the yield increases with bead size. This is due to the dramaticincrease in inertial lift force with particle size. Taking 80 μL/min perrow as an example, the yield is 100% for 20 μm and 10 μm beads. Thisthen drops to 68% for 8 μm beads, 1% for 7 μm beads, and 0% for 6 μmbeads. Third, for any given device, the flow rate per row provides ameans of fine-tuning the critical particle size. For example, toseparate 10 μm particles from ≤7 μm particles, 80 μL/min is the idealflow rate per row. To separate 8 μm particles from ≤6 μm particles, 150μL/min is the ideal flow rate per row.

In the second experiment, the impact of fluid shift on the yield of WBCswas evaluated. WBCs were isolated using hetastarch sedimentation.Specifically, 1 mL of 6% hetastarch (Stemcell Technologies HetaSep) wasadded to 10 mL of fresh whole blood, mixed, and left to sediment for 30minutes. The top, WBC-enriched (and RBC-depleted) layer was thenaspirated with a pipette. This sample was introduced into one of sixdifferent devices, each of which had a different shift (2.5%, 3.0%,3.2%, 3.4%, 3.6%, or 4.0%) and dimensions as described above. In eachcase, the sample entered the device alongside a stream of buffer. Thetotal flow rate (sample+buffer) was chosen to give 80 μL/min flow rateper row, and the relative input flow rates of the sample and buffer werechosen such that 18% of the total input flow was sample. At the end ofthe device, WBCs that had migrated downward (across the array) exitedthe device through the product channel and were collected in a vial.WBCs that remained at the top of the array exited the device through thewaste channel and were collected in a separate vial. The volumes of theproduct and waste vials were measured by mass, and the concentrations ofthe WBCs were determined using standard Neubauer and Nageotte countingchambers. The relative yield was calculated as the fraction of outputWBCs in the product.

FIG. 12 is a plot that shows the dependence of WBC yield on fluid shift.From 2.5% shift to 3.2% shift, the yield drops from 96% to 93%. Beyond3.2% shift, the drop in yield steepens, falling to 71% at 4.0% shift.This indicates that for the smaller shifts tested, the migration of WBCsdue to inertial lift forces is large enough for essentially all of theWBCs to escape the fluid that shifts between islands. However, for thelarger shifts, some WBCs, presumably the smaller WBCs, are unable toescape the fluid that shifts between islands and thereby end up in thewaste

Multiplexed Devices

In some implementations, island arrays, such as those described herein,can be multiplexed to create very high throughput devices because thefootprint of each array is small. FIG. 13 is an image of a standardmicroscope slide (25 mm×75 mm) that accommodates 46 arrays operating inparallel and arranged as 23 duplexes. The multiplexed array enables acombined blood sample throughput of up to ˜1.4 mL/min.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims.

What is claimed is:
 1. A method of producing a sample enriched inparticles of a first type, the method comprising: obtaining a fluidsample comprising particles of the first type; altering a size of theparticles of the first type in the fluid sample to provide particles ofthe first type having an increased effective size in the fluid sample;and flowing the fluid sample containing the particles of the first typehaving the increased effective size into a particle sorting region of amicrofluidic device comprising a first outer wall of the particlesorting region; a second outer wall of the particle sorting region; afirst microfluidic channel extending longitudinally along the particlesorting region between the first outer wall and the second outer wall; asecond microfluidic channel extending longitudinally between the firstmicrofluidic channel and the first outer wall; a first array of islandsseparating the first microfluidic channel from the second microfluidicchannel, wherein each island in the first array is separated from anadjacent island in the first array by an opening that fluidly couplesthe first microfluidic channel to the second microfluidic channel; athird microfluidic channel extending longitudinally between the firstmicrofluidic channel and the second outer wall; a second array ofislands separating the first microfluidic channel from the thirdmicrofluidic channel, wherein each island in the second array isseparated from an adjacent island in the second array by an opening thatfluidly couples the first microfluidic channel to the third microfluidicchannel; wherein, over a length of the particle sorting region thatincludes a plurality of islands from the first array and from the secondarray, a distance from the first outer wall to the first array ofislands increases so that a fluidic resistance of the secondmicrofluidic channel decreases along a longitudinal direction of theparticle sorting region relative to the fluidic resistance of the firstmicrofluidic channel, such that a portion of fluid from the fluid samplein the first microfluidic channel passes through the first array intothe second microfluidic channel, and wherein, over the length of theparticle sorting region that includes the plurality of islands from thefirst array and from the second array, a distance from the second outerwall to the second array of islands decreases so that a fluidicresistance of the third microfluidic channel increases along thelongitudinal direction of the particle sorting region relative to thefluidic resistance of the first microfluidic channel, such that aportion of fluid from the fluid sample in the third microfluidic channelpasses through the second array into the first microfluidic channel. 2.The method of claim 1, wherein inertial lift forces cause the particlesof the first type having the increased effective size to remain withinthe first microfluidic channel.
 3. The method of claim 1, whereinaltering the size of the particles of the first type comprises bindingthe particles of the first type to one or more other particles.
 4. Themethod of claim 3, wherein the one or more other particles comprisebeads.
 5. The method of claim 1, wherein altering the size of theparticles of the first type comprises binding the particles of the firsttype to one or more other particles of a second type that is differentfrom the first type.
 6. The method of claim 1, wherein the particles ofthe first type are cells, and wherein altering the size of the particlesof the first type comprises forming cell aggregates.
 7. The method ofclaim 1, wherein the particles of the first type are cells, and whereinaltering the size of the particles of the first type comprises causingosmotic swelling of the cells.
 8. The method of claim 1, furthercomprising focusing the particles of the first type having the increasedeffective size along one or more streamlines within the firstmicrofluidic channel.
 9. The method of claim 2, wherein the fluid samplecomprises particles of a second type having a particle size smaller thanthe effective size of the particles of the first type, and whereininertial lift forces are insufficient to prevent the particles of thesecond type from flowing with the portion of the fluid sample passingthrough one or more of the openings between adjacent islands into thesecond microfluidic channel.
 10. The method of claim 1, wherein across-sectional area of the first microfluidic channel between the firstarray of islands and the second array of islands is substantiallyconstant along a longitudinal direction of the microfluidic device. 11.The method of claim 1, wherein the first array of islands consists of asingle row of islands.
 12. The method of claim 1, wherein the firstarray of islands comprises an array of two or more rows of islands. 13.The method of claim 1, wherein the second array of islands consists of asingle row of islands.
 14. The method of claim 1, wherein the secondarray of islands comprises array of two or more rows of islands.